The work described in this proposal involves the development of novel analytical methods for use in biochemical and medical research as well as in the clinical chemistry laboratory. The work is primarily segmented along two lines namely thermoanalytical methods and chemiluminescent procedures. Thermochemical methods will be used to detect the heat generated when a substrate is converted to a product by passing it over an enzyme immobilized on porous glass. We propose to develop thermochemical sensors for the determination of penicillin and triglycerides. We will also utilize immobilized enzymes in conjunction with a coulometric pH stat to determine the purity of glucose as well as develop a clinical method for measuring glucose in serum via the use of glucose oxidase. Chemiluminescence reactions can detect hydrogen peroxide with high sensitivity. We will use chemiluminescence from the reaction of organic oxalates with hydrogen peroxide in the presence of fluorescer to analyze and galactose and cholesterol using galactose and cholesterol oxidases. Many enzyme systems are coupled to the formation or consumption of the reduced form of NADH. NADH is oxidized to peroxide by oxygen in the presence of a methylene blue. Chemiluminescence will be used to assay the activity of LDH by coupling it to the formation of NADH and ultimately peroxide. Phosphorous can be determined by flame chemiluminescence. By vaporizing a sample in a graphite furnace before introducing it to a flame, it is possible to eliminate interferences affecting emission intensity. We proposes to use this sytem to develop methods for phosphorus in serum and urine. We are investigating three additional aspects of luminescence analysis. First, we are studying the application of statistical methods to the resolution of luminescence intensity-time curves. Second, we are investigating the use of radical ion-peroxide reactions as a luminescence excitation reaction. Third, electrochemiluminescence is being studied for possible utilization as an analytical tool. Bibliographic references: L.M. Canning, Jr. and P.W. Carr, "Rapid Thermochemical Analysis Via Immobilized Enzyme Columns", Anal. Lett., 8, 359 (1975). W.D. Bostick and P.W. Carr, "Feasibility of Recording Clot Lysis with the Thermometric Clot Detector", Clin. Chem., 20, 1435 (1974).